Stroke, defined as a sudden weakening or loss of consciousness, sensation and voluntary motion caused by rupture or obstruction of an artery of the brain, is the third cause of death in the United States. Worldwide, stroke is the number one cause of death due to its particularly high incidence in Asia. Ischemic stroke is the most common form of stroke, being responsible for about 85% of all strokes, whereas hemorrhagic strokes (e.g. intraparenchymal or subarachnoid) account for the remaining 15%. Due to the increasing mean age of the population, the number of strokes is continuously increasing. Because the brain is highly vulnerable to even brief ischemia and recovers poorly, primary prevention in ischemic stroke prevention offers the greatest potential for reducing the incidence of this disease.
Focal ischemic cerebral infarction occurs when the arterial blood flow to a specific region of the brain is reduced below a critical level. Cerebral artery occlusion produces a central acute infarct and surrounding regions of incomplete ischemia (sometimes referred to as ‘penumbra’), that are dysfunctional-yet potentially salvageable. Ischemia of the myocardium, as a result of reduced perfusion due to chronic narrowing of blood vessels, may lead to fatal heart failure and constitutes a major health threat. Acute myocardial infarction, triggered by coronary artery occlusion, produces cell necrosis over a time period of several hours. In the absence of reflow or sufficient perfusion, the cerebral or myocardial ischemic regions undergo progressive metabolic deterioration, culminating in infarction, whereas restoration of perfusion in the penumbra of the brain infarct or in the jeopardized but salvageable region of the myocardium may ameliorate the tissue damage.
Growth factor mediated improved perfusion of the penumbra in the brain or of the jeopardized myocardium of patients suffering ischemic events, either via increased vasodilation or angiogenesis (the formation of endothelial-lined vessels), may be of great therapeutic value according to Isner et al. in J. Clin. Invest. (1999) 103(9):1231-6 but many questions yet remain to be answered in this respect. This is also true for ischemic conditions related to peripheral limbs (eg peripheral arterial disease, peripheral ischemia). For instance, an outstanding question is whether formation of new endothelial-lined vessels (i.e. angiogenesis) alone is sufficient to stimulate sustainable functional tissue perfusion. Indeed, coverage of endothelial-lined vessels by vascular smooth muscle cells (i.e. arteriogenesis) provides vasomotor control, structural strength and integrity and renders new vessels resistant to regression.
Capillary blood vessels consist of endothelial cells and pericytes, which carry all the genetic information required to form tubes, branches and entire capillary networks. Specific angiogenic molecules can initiate this process. A number of polypeptides which stimulate angiogenesis have been purified and characterized as to their molecular, biochemical and biological properties, as reviewed by Klagsbrun et al. in Ann. Rev. Physiol. (1991) 53:217-239 and by Folkman et al. in J. Biol. Chem. (1992) 267:10931-4. One factor that can stimulate angiogenesis and which is highly specific as a mitogen for vascular endothelial cells, is termed vascular endothelial growth factor (hereinafter referred as VEGF) according to Ferrara et al. in J. Cell. Biochem. (1991) 47:211-218. VEGF is also known as vasculotropin. Connolly et al. also describe in J. Biol. Chem. (1989) 264:20017-20024, in J. Clin. Invest. (1989) 84:1470-8 and in J. Cell. Biochem. (1991) 47:219-223 a human vascular permeability factor that stimulates vascular endothelial cells to divide in vitro and promotes the growth of new blood vessels when administered into healing rabbit bone grafts or rat corneas. The term vascular permeability factor (VPF for abbreviation) was adopted because of increased fluid leakage from blood vessels following intradermal injection and appears to designate the same substance as VEGF. The murine VEGF gene has been characterized and its expression pattern in embryogenesis has been analyzed. A persistent expression of VEGF was observed in epithelial cells adjacent to fenestrated endothelium, e.g. in chloroid plexus and kidney glomeruli, which is consistent with its role as a multifunctional regulator of endothelial cell growth and differentiation as disclosed by Breier et al. in Development (1992) 114:521-532. VEGF shares about 22% sequence identity, including a complete conservation of eight cysteine residues, according to Leung et al. in Science (1989) 246:1306-9, with human platelet-derived growth factor PDGF, a major growth factor for connective tissue. Alternatively spliced mRNAs have been identified for both VEGF and PDGF and these splicing products differ in their biological activity and receptor-binding specificity. VEGF is a potent vasoactive protein that has been detected in and purified from media conditioned by a number of cell lines including pituitary cells, such as bovine pituitary follicular cells (as disclosed by Ferrara et al. in Biochem. Biophys. Res. Comm. (1989) 161:851-858 and by Gospodarowicz et al. in Proc. Natl. Acad. Sci. USA (1989) 86: 7311-5), rat glioma cells (as disclosed by Conn. et al. in Proc. Natl. Acad. Sci. USA (1990) 87:1323-1327) and several tumor cell lines. Similarly, an endothelial growth factor isolated from mouse neuroblastoma cell line NB41 with an unreduced molecular mass of 43-51 kDa has been described by Levy et al. in Growth Factors (1989) 2:9-19.
Recent data show that VEGF has a modest effect on collateral growth and limb perfusion measured by microspheres (Deindi et al. in Circ. Res. (2001) 89:779-86), but increases perfusion measured by laser doppler (Isner et al. in Circulation (1999) 99:3188-98). Thus, despite a slight increase in collateral growth and hind limb perfusion, the overall functional reserve after VEGF treatment was impaired, presumably because of unfavorable hemodynamic effects. In other words, it is known that despite vessel formation by means of VEGF, the functionality of the vessels formed is very low. Despite promising initial clinical trials, a large double-blind placebo-controlled trial testing the efficacy of intracoronary and intravenous VEGF for therapeutic angiogenesis in patients with chronic myocardial ischemia not amenable to standard revascularization techniques, indicated that even high dose treatment with VEGF did not lead to a significant improvement in ETT time (Henry et al. 2003, Circulation, 107:1359-1365).
Placental growth factor (hereinafter referred as PIGF) was first disclosed by Maglione et al. in Proc. Natl. Acad. Sci. USA (1991) 88(20):9267-71 as a protein related to the vascular permeability factor. U.S. Pat. No. 5,919,899 discloses nucleotide sequences coding for a protein, named PIGF, which can be used in the treatment of inflammatory diseases and in the treatment of wounds or tissues after surgical operations, transplantations, burns of ulcers and so on. Soluble non-heparin binding and heparin binding forms, built up of 131 and 152 amino-acids and known as PIGF-1 and PIGF-2 respectively, have been described for PIGF and were found to be differentially expressed in placenta, trophoblastic tumors and cultured human endothelial cells (Maglione et al., 1993).
Carmeliet et al. Nat. Med. (2001) 7:575-583 provides an extensive and comprehensive review of the state of the art related to PIGF as follows. VEGF stimulates angiogenesis by activating the VEGF tyrosine kinase receptor-2 (VEGFR-2). Recombinant PIGF stimulates angiogenesis in particular conditions and induces vascular permeability when co-injected with VEGF, but the role of endogenous PIGF remains unknown. Both PIGF and VEGF bind to VEGF receptor-1 (VEGFR-1). PIGF has been proposed to stimulate angiogenesis by displacing VEGF from the ‘VEGFR-1 sink’, thereby increasing the fraction of VEGF available to activate VEGFR-2. Alternatively, PIGF might stimulate angiogenesis by transmitting intracellular signals through VEGFR-1. PIGF might also affect angiogenesis by forming heterodimers with VEGF, but their role is controversial.
The role of PIGF in angiogenesis was evaluated by inactivating the gene expressing PIGF (Pgf) in mice. Unexpectedly, the absence of PIGF had a negligible effect on vascular development. However, PIGF deficiency reduced pathological angiogenesis, permeability and collateral growth in ischemia, inflammation and cancer.
Carmeliet et al. (cited supra) studied the growth of collateral arteries after ligation of the femoral artery in mice and found that PIGF levels were 45% higher in ligated vessels than in control vessels. On the other hand macrophages, known to play an essential role in collateral growth, infiltrated significantly more collaterals in wild-type mice than in Pgf−/− mice. In another experiment, mice were transplanted with congenic bone marrow and capillary ingrowth in matrigel, supplemented with the VEGF165 isoform, and angiogenesis was quantified by measuring the hemoglobin content per implant. Matrigel angiogenesis was abundant when both the donor bone marrow and the host vessels produced PIGF but minimal when the bone marrow and host lacked PIGF. Capillaries still infiltrated the matrigel in wild-type mice transplanted with Pgf−/− bone marrow, indicating that PIGF production by vessel-wall-associated endothelial cells was sufficient for angiogenesis. When a Pgf−/− recipient was transplanted with wild-type bone marrow, matrigel angiogenesis still occurred, indicating that production of PIGF by bone-marrow cells can stimulate angiogenesis at distant sites. Transplantation of wild-type bone marrow in Pgf−/− mice also rescued the impaired collateral enlargement after ligation of the femoral artery, possibly by mobilizing PIGF-producing monocytes/macrophages to the collaterals. In still another experiment, in order to determine whether the amplification of the VEGF response by PIGF resulted from direct stimulation of endothelial cells, Carmeliet et al. (cited supra) studied capillary outgrowth in intact aortic rings and found that, because PIGF alone (in the absence of VEGF) did not stimulate isolated endothelial cells, PIGF probably stimulated capillary outgrowth by amplifying endogenous VEGF in aortic rings. Carmeliet et al. (cited supra) further observed that even at low doses PIGF dose-dependently restored the impaired VEGF response of Pgf−/− endothelial cells. Further, PIGF activated VEGFR-1. These findings altogether indicate that PIGF, via activation of VEGFR-1, specifically potentiates the angiogenic response to VEGF and that by affecting vascular growth and remodeling, PIGF contributes to the pathogenesis of several disorders with high morbidity.